The characteristic amyloid lesions of Alzheimer's disease (AD) are primarily composed of Amyloid β (Aβ) (Glenner & Wong, 1984), a 39-43 amino acid protein which is a normally soluble protein found in biological fluids. Amyloid formation is linked to the pathogenesis of the disease, so identifying the neurochemical changes which lead to the inhibition of Aβ catabolism and its accumulation in the neocortex would be an important clue to the pathogenesis of AD.
Although the fundamental pathology, genetic susceptibility and biology associated with AD are becoming clearer, a rational chemical and structural basis for developing effective drugs to prevent or cure the disease remains elusive. While the genetics of AD indicate that the metabolism of Aβ is intimately associated with the pathogenesis of the disease as indicated above, drugs for the treatment of AD have so far focused on “cognition enhancers”, which do not address the underlying disease processes. These drugs have met with only limited success.
The nature of the deranged neurochemical environment in AD can be partly deduced from the post-translational modifications of amyloid Aβ. Aβ extracted from biological systems normally migrates as an apparent ˜4 kD monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; (Shoji et al., 1992)); however, Aβ extracted from specimens of AD-affected post-mortem brain migrates on SDS-PAGE as SDS-, urea- and formic acid-resistant oligomers (Masters et al., 1985; Roher et al., 1996; Cherny et al., 1999).
Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) of these SDS-resistant oligomers extracted from neuritic plaque and vascular amyloid indicates the presence of covalently cross-linked dimeric and trimeric Aβ species (Roher et al., 1996).
Synthetic Aβ1-40 and Aβ1-42 normally migrate as apparent monomers on SDS-PAGE, but form apparent higher molecular weight species upon incubation (Burdick et al., 1992). This process is accelerated by exposure to oxidative systems (Dyrks et al., 1992; Atwood et al., 1997).
Tyrosine cross-linking has been proposed as a mechanism of Aβ oligomerization in vivo, since tyrosine residues in synthetic human Aβ can be cross-linked by peroxidase-catalyzed oxidation systems (Galeazzi et al., 1999). As Rat Aβ, unlike human Aβ, lacks a tyrosine residue (Atwood et al., 1997), it is therefore resistant to metal-catalyzed oxidative oligomerization, and this perhaps explains the rarity of amyloid deposits in these animals (Vaughan and Peters, 1981).
Tyrosine cross-linking in proteins is a sensitive marker of oxidative stress. Covalent carbon-carbon bridges or carbon-oxygen bridges are formed between single tyrosyl residues and/or dityrosyl residues, resulting in a number of stable, fluorescent reaction products (Gross and Sizer, 1959; Amado et al., 1984, Jacob et al., 1996). The major reaction products of the free tyrosyl radical are the intensely fluorescent amino acids 3,3′-dityrosine (DT), 3,3′,3′-trityrosine (TT) and pulcherosine (P), and the non-fluorescent isodityrosine (iso-DT) (Gross and Sizer, 1959; Amado et al., 1984, Jacob et al., 1996; Heinecke et al., 1993). DT and 3-nitrotyrosine levels are elevated in the hippocampus and neocortical of brains of patients with AD compared to the same regions of normal brain, and are also elevated in ventricular cerebrospinal fluid in AD patients (Hensley et al., 1998).
Tyrosine cross-linking may also be important in other neurodegenerative diseases such as Parkinson's disease, and other conditions in which α-synuclein fibrils are deposited. These include Parkinson's disease itself, dementia with Lewy body formation, multiple system atrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease. Exposure of recombinant α-synuclein to nitrating agents results in nitration of tyrosine residues as well as oxidation of tyrosine to form DT; this results in cross-linking of α-synuclein to form stable aggregates (Souza et al, 2000). The same authors also found that monoclonal antibodies raised against nitrated synuclein bound specifically to Lewy bodies and to glial cell inclusions in a variety of synucleinopathies (Duda et al., in preparation referred to in Souza et al., 2000).
We have now found that human amyloid-derived Aβ contains tyrosine cross-links, and includes both dityrosine and trityrosine cross-linked species. These cross-links can be replicated in vitro, for example by incubating synthetic human Aβ with peroxidase and H2O2, or with H2O2 in the presence of copper ions. These modifications are protease-resistant, and therefore we propose that tyrosine cross-linkage in AD caused by abnormal interaction of Aβ with H2O2 and peroxidases or copper ions contributes to the formation of neurotoxic Aβ oligomers, and to the deposition of Aβ. Immunization against low molecular weight tyrosine cross-linked compounds rather than with whole Aβ can therefore be used for treatment or prevention of AD, without the risk of provoking autoimmune complications which could otherwise be induced by immunization with intact Aβ or large fragments thereof. By restricting the target for immunotherapy to an abnormal fragment or portion of the molecule, it may be possible to minimise undesirable interference with the normal function of the molecule, while providing an active therapy against the abnormal molecule. It will be appreciated that either active or passive immunization may be used.
The oxidative processes which give rise to covalent cross-linking of proteins via tyrosine are also associated with other disorders which are characterised by pathological aggregation and accumulation of specific proteins. It is therefore considered that these conditions also will be amenable to prevention or treatment by the method of the invention.
It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in Australia or in any other country.